Hematopoietic stem cell expansion method
11702635 · 2023-07-18
Assignee
Inventors
Cpc classification
C12N2501/999
CHEMISTRY; METALLURGY
International classification
Abstract
A method to expand hematopoietic stem and progenitor cells (HSPC) wherein the method comprises obtaining an isolated population of HSPC the culturing the isolated population of HSPC in the presence of a histone deacetylase inhibitor (HDAC inhibitor), to form a cultured population, then adding an aminothiol compound to the cultured population.
Claims
1. A method to expand hematopoietic stem and progenitor cells (HSPC) wherein the method comprises; i) obtaining an isolated population of HSPC ii) culturing the isolated population of HSPC in the presence of a histone deacetylase inhibitor (HDAC inhibitor), to form a cultured population iii) adding an aminothiol compound having the formula RNH(C.sub.nH.sub.2n)NH(C.sub.nH.sub.2n)SX, wherein R is hydrogen, an aryl, an acyl, or an alkyl group containing from 1 to 7 carbon atoms, each n has a value of from 2 to 6 and X is H or PO.sub.3H.sub.2; or a pharmaceutically acceptable salt thereof, to the cultured population of HSPC to form expanded cells.
2. The method of claim 1, wherein step i) comprises selecting for cells which are CD133+.
3. The method of claim, wherein step i) comprises selecting for cells which are CD34+.
4. The method of claim 1, wherein the HDAC inhibitor is scriptaid or quisinostat.
5. The method of claim 1, wherein the HDAC inhibitor is scriptaid.
6. The method of claim 1, wherein the aminothiol compound is amifostine: ##STR00004## or WR1065: ##STR00005##
7. The method of claim 1, wherein the aminothiol compound is WR1065.
8. The method of claim 1, wherein the isolated population is obtained from umbilical cord blood.
9. The method of claim 1, wherein the isolated population is obtained from peripheral blood.
10. The method of claim 1, wherein the isolated population obtained from bone marrow.
11. The method of claim 1, wherein the steps ii) and iii) are carried out over a total time of 5 to 10 days to form expanded cells.
12. The method of claim 1, wherein the cells are cultured in a serum free or feeder free tissue culture system.
13. The method of claim 1, wherein the cells are obtained from a mammal, preferably a human.
14. The method of claim 1, wherein the expanded cells are enriched for HSC.
15. The method of claim 1, wherein the expanded cells are enriched for; Lin−, CD38−, CD34+, CD133+, CD45RA−, CD90+ and CD49f+.
16. The method of claim 1, wherein the total cell expansion is between about 2-fold to about 20-fold.
17. The method of claim 1, wherein the expansion of Lin−, CD38−, CD34+, CD133+, CD45RA−, CD90+ and CD49f+ cells is about 500-fold.
18. The method of claim 1, wherein the HDAC inhibitor is used at a concentration of 0.01-50 μM.
19. The method of claim 1, wherein the aminothiol compound is used at a concentration of 50-500 μM.
Description
BRIEF DESCRIPTION OF THE FIGURES
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DETAILED DESCRIPTION OF THE INVENTION
(15) As used herein the term hematopoietic stem and progenitor cells (HSPC) refers to cells found in bone marrow, umbilical cord blood and peripheral blood which can differentiate and/or proliferate to form blood cells, examples of blood cells include, but is not restricted to, monocytes, macrophages, neutrophils, basophils, eosinophils, erythrocytes, dendritic cells, megakaryocytes, platelets, T cells, B cells, and natural killer cells.
(16) As used herein the term “hematopoietic stem cells” or “HSC” refers to multipotent or pluripotent cells which have the ability to differentiate into blood cells of all lineages and to regenerate themselves whilst maintaining their pluripotent characteristics. The term “HSC” as used herein refers to cells which are; Lin−, CD38−, CD34+, CD133+, CD45RA−, CD90+ and CD49f+. Within the terms “CD34+”, “CD133+”, “CD90+”, “CD49f+” the (+) designation indicates that the specified cluster of differentiation (CD) is expressed by the cell and is present on the cell surface. Within the terms “CD38−”, “CD45RA−” the (−) designation indicates that the specified CD is not expressed or poorly expressed by the cell. However, human embryonic stem cells and any cell resulting from the destruction of a human embryo are not within the scope of the invention.
(17) As used herein the term “isolated population” refers to a sample of cells which has been obtained from a source. Wherein the cells may have been obtained commercially, or wherein the cells were obtained from a subject. The source of an isolated population includes, but is not restricted to, umbilical cord blood, bone marrow and peripheral blood. The “isolated population” may have been obtained from a source which is fresh or frozen, wherein a fresh source has not been frozen prior to use. If the sample is frozen then the cells will be thawed before use in the method.
(18) As used herein the term “cultured population” refers to an isolated population of cells which has been propagated in an artificial medium ex vivo. It will be obvious to a skilled person what type of artificial media to use, an example of a suitable media is StemSpan ACF media (Stem Cell Technologies). The artificial media may also be supplemented with other factors or cytokines to improve the growth of the cells, examples of supplements include, but are not restricted to, stem cell factor (SCF), fms-related tyrosine kinase 3-ligand (FLT3LG) and thrombopoietin (TPO). The isolated population can be cultured/propagated over a number of days to form a cultured population. In some embodiments, the culturing time is from 2 to 20 days, more preferably 3 to 20, most preferably 4 to 15 days. For example the culturing/propagating can take place over 20, 15, 10, 9, 8, 7, 6, 5 or 4 days. The total culturing time encompasses the time taken to perform steps (ii) and (iii).
(19) The term histone deacetylase inhibitor (HDAC inhibitor) as used herein refers to a compound which inhibits the activity of the enzyme histone deacetylase. There are four classifications of histone deacetylase; class I, class II, class III, and class IV. Based on their sequence homology and domain organisation, class II inhibitors can be further subdivided into class IIa and class IIb. As used herein the term histone deacetylase refers to compound which can inhibit the activity of any of the classes of histone deacetylase.
(20) Examples of HDAC inhibitors include, but are not restricted to, Scriptaid, Vorinostat, Tacedinaline, RG2833, RGFP966, Trichostatin A, LMK235, Tubastatin A, Quisinostat, LBH589, PXD101, ITF2357, PCI-24781, FK228 MS-275, MGCD0103, Sodium Phenylbutyrate, Valproic acid, AN-9, Baceca, Savicol.
(21) An aspect of the invention includes the use of an aminothiol compound having the formula RNH(C.sub.nH.sub.2n)NH(C.sub.nH.sub.2n)SX, wherein R is hydrogen, an aryl, an acyl, or an alkyl group containing from 1 to 7 carbon atoms, each n has a value of from 2 to 6 and X is H or PO.sub.3H.sub.2; or a pharmaceutically acceptable salt thereof.
(22) As used herein, “aryl” means a monocyclic, bicyclic, or tricyclic monovalent or divalent (as appropriate) aromatic radical, such as phenyl, biphenyl, naphthyl, anthracenyl, which can be optionally substituted with up to five substituents preferably selected from the group of C.sub.1-C.sub.6 alkyl, hydroxy, C.sub.1-C.sub.3 hydroxyalkyl, C.sub.1-C.sub.3 alkoxy, C.sub.1-C.sub.3 haloalkoxy, amino, C.sub.1-C.sub.3 mono alkylamino, C.sub.1-C.sub.3 bis alkylamino, C.sub.1-C.sub.3 acylamino, C.sub.1-C.sub.3 aminoalkyl, mono (C.sub.1-C.sub.3 alkyl) amino C.sub.1-C.sub.3 alkyl, bis(C.sub.1-C.sub.3 alkyl) amino C.sub.1-C.sub.3 alkyl, C.sub.1-C.sub.3-acylamino, C.sub.1-C.sub.3 alkyl sulfonylamino, halo, nitro, cyano, trifluoromethyl, carboxy, C.sub.1-C.sub.3 alkoxycarbonyl, aminocarbonyl, mono C.sub.1-C.sub.3 alkyl aminocarbonyl, bis C.sub.1-C.sub.3 alkyl aminocarbonyl, —SO.sub.3H, C.sub.1-C.sub.3 alkylsulfonyl, aminosulfonyl, mono C.sub.1-C.sub.3 alkyl aminosulfonyl and bis C.sub.1-C.sub.3-alkyl aminosulfonyl.
(23) As used herein, “alkyl” means a C.sub.1-C.sub.7 alkyl group, which can be linear or branched. Preferably, it is a C.sub.1-C.sub.6 alkyl moiety. More preferably, it is a C.sub.1-C.sub.4 alkyl moiety. Examples include methyl, ethyl, n-propyl and t-butyl. It may be divalent, e.g. propylene.
(24) As used herein, acyl is an alkyl group as defined above, which includes a carbonyl group (C═O).
(25) Each of the alkyl and acyl groups may be optionally substituted with aryl, cycloalkyl (preferably C.sub.3-C.sub.10) or heteroaryl. They may also be substituted with halogen (e.g. F, Cl), NH.sub.2, NO.sub.2 or hydroxyl.
(26) As used herein the term “umbilical cord blood” has its conventional use in the art; that is generally the blood that is left in the umbilical cord and placenta post-partum. Human cord blood is within the scope of the present invention and is obtained with written informed pre-consent and ethical approval.
(27) As used herein the term “peripheral blood” has its conventional use in the art; that is generally blood which is circulating throughout the circulatory system. Human peripheral blood is within the scope of the present invention and is obtained with written informed pre-consent and ethical approval.
(28) As used herein the term “bone marrow” has its conventional use in the art; that is, generally the gelatinous tissue present in bone cavities. The tissue comprises red bone marrow, a subset of bone marrow having populations of hematopoietic stem cells, progenitor cells and precursor cells. Human bone marrow is within the scope of the present invention and is obtained with written informed pre-consent and ethical approval.
(29) As used herein the term “expanded cells” refers to cells which have been cultured ex vivo, under appropriate conditions, and undergone cell division to amplify the number of cells. As used herein the term “cell expansion” refers to the amplification of the number of cells by the ex vivo culturing of cells under appropriate conditions, wherein the number of cells present at the end of culturing is greater than the number of cells present at the start of culturing.
(30) Within the cells, wherein the cells may be part of the isolated population of cells, the cultured population of cells or the expanded cells, there are subtypes of cells. Examples of the cell subtypes are, but not restricted to; hematopoietic stem cells, hematopoietic progenitor cells and cells as defined by their phenotypic markers. Non-limiting examples of phenotypic markers are; Lin or CD38 or CD34 or CD133 or CD45RA or CD90 or CD49f, wherein the cells can also be defined by combinations of these phenotypic markers. As used herein the term “enriched” is used to refer to a set of cells which contains a high proportion of a specific subset/subtype of cell, wherein the set of cells may contain 2%, or 5%, or 10%, or 15%, or 20%, or 25%, or 30%, or 35%, or 40%, or 45%, or 50%, or 55%, or 60%, or 65%, or 70%, or 75%, or 80%, or 85%, or 90% of the specific subset/subtype of cell. Within the present invention the term “enriched” can be used to refer to a population of cells wherein the cells have undergone expansion and wherein a specific subtype of cells have increased in number proportionally more than other cells within the population. This enriched population of cells contains a significant proportion of a specific subtype of cells, wherein the significant proportion may be 2%, or 5%, or 10%, or 15%, or 20%, or 25%, or 30%, or 35%, or 40%, or 45%, or 50%, or 55%, or 60%, or 65%, or 70%, or 75%, or 80%, or 85%, or 90% of the total population.
(31) As used herein the term “serum free tissue culture system” refers to culturing cells in a media which has not been supplemented with serum derived from an animal.
(32) As used herein the term “feeder free tissue culture system” refers to a method of culturing cells without utilising a layer of connective tissue cells to support and provide metabolites to the growing cells.
(33) As used herein the term “total cell expansion” refers to the increase in number of total nucleated cells.
(34) As used herein the term “total culturing time” refers to the time in which steps ii) and iii) are carried out. Wherein step ii) comprises culturing the isolated population of HSPC in the presence of a histone deacetylase inhibitor (HDAC inhibitor), to form a cultured population, and step iii) comprises adding an aminothiol compound having the formula RNH(C.sub.nH.sub.2n)NH(C.sub.nH.sub.2n)SX, wherein R is hydrogen, an aryl, an acyl, or an alkyl group containing from 1 to 7 carbon atoms, each n has a value of from 2 to 6 and X is H or PO.sub.3H.sub.2; or a pharmaceutically acceptable salt thereof, to the cultured population of HSPC to form expanded cells. During the total culturing time the cells are allowed to grow on an appropriate media supplemented with a HDAC inhibitor, the end of the total culturing time is signified by the cells being harvested/pooled/analysed.
(35) An aspect of the present invention is a method to expand hematopoietic stem and progenitor cells (HSPC) wherein the method comprises;
(36) i) obtaining an isolated population of HSPC
(37) ii) culturing the isolated population of HSPC in the presence of a histone deacetylase inhibitor (HDAC inhibitor), to form a cultured population
(38) iii) adding an aminothiol compound having the formula RNH(C.sub.nH.sub.2n)NH(C.sub.nH.sub.2n)SX, wherein R is hydrogen, an aryl, an acyl, or an alkyl group containing from 1 to 7 carbon atoms, each n has a value of from 2 to 6 and X is H or PO.sub.3H.sub.2; or a pharmaceutically acceptable salt thereof, to the cultured population of HSPC to form expanded cells.
(39) In an embodiment of the present invention, step i) further comprises selecting for cells which are CD133+. In some embodiments, the isolated population comprises cells which are CD133+. In an embodiment step i) further comprises selecting for cells which are CD34+. If the isolated population of HSPC is obtained from a source that has been frozen it may be preferable to select for cells which are CD34+. If the isolated population of HSPC is obtained from a fresh source then it may be preferable to select for cells which are CD133+. Suitable methods for selecting cells by cell surface markers are known in the art for example using Magnetic Activated Cell Sorting (MACs) or Fluorescent Activated Cell Sorting (FACS). Preferably, the isolated population comprises cells which are CD38− or CD34+ or CD133+ or CD45RA− or CD90+ or CD49f+, or any combination thereof.
(40) In one embodiment of the present invention, the isolated population of cells is obtained from umbilical cord blood or bone marrow or peripheral blood. In a preferred embodiment, the isolated population of cells is obtained from umbilical cord blood. In some embodiments, the cells are obtained from a mammal (for example mouse, rat, dog or human). A preferred embodiment is wherein the cells are obtained from a human.
(41) In one embodiment of the present invention the HDAC inhibitor is selected from a broad-spectrum inhibitor, or a selective class I, class IIa, class IIb, class III or class IV inhibitor. Preferably, the HDAC inhibitor is selected from a broad-spectrum inhibitor, or a selective class I, class IIa, class III or class IV inhibitor. More preferably, the HDAC inhibitor is a broad-spectrum inhibitor, class I or class IIa inhibitor.
(42) In a preferred embodiment, the HDAC inhibitor is selected from scriptaid, RG2833, RGFP966, LMK235, Tubastatin A, quisinostat, sodium phenylbutyrate. In some embodiments of the present invention, the HDAC inhibitor is a scriptaid or quisinostat. In a preferred embodiment the HDAC inhibitor is scriptaid, which has the structure;
(43) ##STR00001##
(44) An aminothiol compound of the invention has the formula RNH(C.sub.nH.sub.2n)NH(C.sub.nH.sub.2n)SX, wherein R is hydrogen, an aryl, an acyl, or an alkyl group containing from 1 to 7 carbon atoms, each n has a value of from 2 to 6 and X is H or PO.sub.3H.sub.2; or a pharmaceutically acceptable salt thereof.
(45) In a preferred embodiment, R is hydrogen.
(46) In some embodiments, the aminothiol compound of the invention is amofostine:
(47) ##STR00002##
Or
WR1065:
(48) ##STR00003##
(49) Most preferably, the aminothiol compound is WR1065.
(50) In one embodiment of the present invention the HDAC inhibitor is used at a concentration of between 0.01 μM to 50 μM, preferably between 0.1 μM to 10 μM, more preferably, the HDAC inhibitor is used at a concentration of 1 μM.
(51) In a preferred embodiment of the present invention the aminothiol compound e.g. WR1065, is used at a concentration of 50 μM to 500 μM, preferably 50 μM to 150 μM, more preferably at a concentration of 100 μM.
(52) In a preferred embodiment of the present invention, the HDAC inhibitor is used at a concentration of 1 μM, and preferably the aminothiol compound e.g. WR1065 is used at a concentration of 100 μM.
(53) In some embodiments of the present invention, steps ii) and iii) are performed over two to ten days. In a preferred embodiment steps ii) and iii) are performed over five to 10 days. In a more preferred embodiment steps ii) and iii) are performed over about five days. In an embodiment of the present invention, step iii) begins up to 48 hours before the end of the total culturing time (i.e. the end of step (iii)). In some embodiments of the present invention step iii) begins 16 to 20 hours before the end of the total culturing time. Preferably, steps ii) and iii) are performed over five days and, more preferably, step iii) is performed 16 to 20 hours before the end of the total culturing time.
(54) Preferably, in the present invention, step ii) is performed over 4 to 10 days. In a preferred embodiment, step ii) is performed over at least 4 days. In some embodiments step iii) is performed after the cells have been cultured with the HDAC inhibitor (according to step ii)) for at least 4 to 10 days, preferably at least 4 days. In some embodiments step iii) is performed after the cells have been cultured as in step ii) for at least 4 to 10 days, preferably at least 4 days. Preferably, step iii) is begins up to 48 hours before the end of the total culturing time.
(55) Preferably, the cells are cultured in a serum free tissue culture system. In some embodiments of the present invention, cells are cultured in a feeder free tissue culture system. Whilst the culture system is serum and/or feeder free, various nutrients may be added to provide adequate growth and expansion conditions for cells. Examples of suitable media include, but are not limited to StemSpan ACF media (Stem Cell Technologies), StemPro34 serum-free medium (Invitrogen), Stemline II (Thermo Fisher), HPC Expansion Medium DXF (PromoCell), QBSF-60 (Quality Biological), StemMACS HSC expansion media XF (Miltenyi Biotec). In a preferred embodiment of the present invention the cells are cultured in StemSpan ACF media (Stem Cell Technologies). Suitable media may also contain various additives and components which may be chemical or biological components. These components may be incorporated into the suitable media singly or in combination and the skilled person will be able to choose suitable components as required. These components may also be incorporated during culture as required. Examples of components both biological and chemical include, but are not restricted to; amino acids, vitamins, cytokines, growth factors, hormones, antibiotics, fatty acids, saccharides, sodium, calcium, potassium, magnesium, phosphorus, agar, agarose, methylcellulose, collagen, insulin, transferrin, lactoferrin, cholesterol, ethanolamine, sodium pyruvate, 2-mercaptoethanol, polyethylene glycol, sodium selenite.
(56) Various cytokines may be incorporated into the media and/or incorporated during culture, examples of suitable cytokines include, but are not restricted to; interleukin-1 (IL-1), interleukin-2 (IL-2), interleukin-3 (IL-3), interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-6 (IL-6), interleukin-7 (IL-7), interleukin-8 (IL-8), interleukin-9 (IL-9), interleukin-10 (IL-10), interleukin-11 (IL-11), interleukin-12 (IL-12), interleukin-13 (IL-13), interleukin-14 (IL-14), interleukin-15 (IL-15), interleukin-18 (IL-18), interleukin-21 (IL-21), interferon-α (INF-α), interferon-β (INF-β), interferon-γ (INF-γ), granulocyte-macrophage colony stimulating factor (GM-CSF), stem cell factor (SCF), Wnt1, bone morphogenetic protein 7 (BMP7), angiopoietin-like 5 (ANGPTLS), insulin growth factor binding protein 2 (IGFBP2), erythropoietin (EPO), thrombopoietin (TPO), Fms-like tyrosine kinase 3-ligand (FLT3LG). In an embodiment of the present invention the media is supplemented with SCF, TPO and FLT3LG.
(57) Various growth factors may be incorporated into the media and/or incorporated during culture. Examples of suitable growth factors include, but are not restricted to insulin-like growth factor (IGF), epidermal growth factor (EGF), human epidermal growth factor (hEGF), platelet-derived growth factor (PDGF), fibroblast growth factor 1 (FGF1), nerve growth factor (NGF), macrophage inflammatory protein 1-α (MIP-1α), leukaemia inhibitory factor (LIF).
(58) In an embodiment of the present invention, the isolated population is cultured at a temperature between 32° C. to 39° C., preferably between 36° C. to 38° C. In an embodiment of the invention the cells are cultured in a humidified incubator with between about 1% to about 50% CO.sub.2, preferably between about 1% to about 25% CO.sub.2, more preferably between about 1% to about 10% CO.sub.2. The present invention can be performed in a culture vessel suitable for animal cell culture. In one embodiment the present invention is performed in Nanex Hematopoietic Stem/Progenitor Cell (HSPC) Expansion Plates or TC treated Corning 24 well plates or suspension Greiner Bio 24 well plates. In a preferred embodiment, the present invention is performed in a conventional cell culture plate or a suitable closed system such as a cell culture bag (e.g VueLife®) or a stirred bioreactor.
(59) In a preferred embodiment of the present invention, the total cell expansion is between about 2-fold to about 50-fold, or from about 2-fold to about 25-fold, or from about 2-fold to about 20-fold. The total cell expansion is determined by measuring the number of total nucleated cells at the start of the culturing time and comparing to the number of total nucleated cells present at the end of the culturing time.
(60) In a preferred embodiment, the expansion of Lin−, CD38−, CD34+, CD133+, CD45RA−, CD90+ and CD49f+ cells is 50 to 800-fold, more preferably 400 to 600-fold, most preferably 500-fold. The expansion of Lin−, CD38−, CD34+, CD133+, CD45RA−, CD90+ and CD49f+ cells is determined by measuring the number of Lin−, CD38−, CD34+, CD133+, CD45RA−, CD90+ and CD49f+ cells present at the start of the culturing time and comparing it to the number of Lin−, CD38−, CD34+, CD133+, CD45RA−, CD90+ and CD49f+ cells present at the end of the culturing time. Suitable methods for determining cell expansion are known in the art and include, for example, multicolour flow cytometric analysis combined with total cell counting, use of absolute counting beads in combination with flow cytometric analysis, cell counts based on imaging analysis of a cell aliquot using a manual or automated hemocytometer (Viacell, Countess, Nucleocounter, Nexcelome)
(61) In some embodiments, the expanded cells are enriched for HSC. In one embodiment the expanded cells are enriched for Lin− or CD38− or CD34+ or CD133+ or CD45RA− or CD90+ or CD49f+ or any combination thereof, preferably wherein the cells are enriched for CD34+, CD133+, more preferably wherein the expanded cells are enriched for CD38−, CD34+, CD133+, most preferably wherein the expanded cells are enriched for Lin−, CD38−, CD34+, CD133+, CD45RA−, CD90+, CD49f+.
(62) An aspect of the present invention is a kit for the expansion of HSPC as defined above, wherein the kit comprises; sterile elements for the expansion of HSPC, a HDAC inhibitor and an aminothiol compound having the formula RNH(C.sub.nH.sub.2n)NH(C.sub.nH.sub.2n)SX, wherein R is hydrogen, an aryl, an acyl, or an alkyl group containing from 1 to 7 carbon atoms, each n has a value of from 2 to 6 and X is H or PO.sub.3H.sub.2; or a pharmaceutically acceptable salt thereof. In a preferred embodiment the kit also contains apparatus and/or materials for obtaining an isolated population of HSPC. A person skilled in the art will know of suitable elements, apparatus and/or materials. Examples include magnetic bead isolation, MACS bead isolation columns, CliniMACS (Miltenyi) or FACS sorting.
(63) The following examples illustrate the invention.
EXAMPLES
Example 1 Expansion of Cells from Umbilical Cord Blood
(64) Materials and Methods
(65) Cell Culture
(66) Human UCB units were collected with written informed pre-consent and ethical approval from Oxford and Berkshire National Research Ethical Committees and studies conducted with approval of the NHSBT research committee. UCB mononuclear cells (MNC) were isolated by density gradient centrifugation on lymphocyte separation medium 1077 (PAA Laboratories, Pasching, Austria; density<1.077 g/ml). CD133+ cells were isolated from the MNC with immunomagnetic beads (Miltenyi Biotec, Germany). After isolation, the cells were cryopreserved in 10% DMSO in FCS (fetal calf serum) and stored at −150° C. in aliquots of 1×10.sup.5 cells/vial. Cells were analysed for the purity of the isolation with flow cytometry on a BD LSR II (BD Biosciences, CA) using CD34-APC, CD133-PE and the appropriate isotype controls (all Miltenyi Biotec). Bone marrow derived CD133+ cells were obtained commercially from Lonza and peripheral blood CD133+ cells were isolated using immunomagnetic beads as above from total peripheral blood mononuclear cells obtained commercially from Lonza Biologics.
(67) Vials from 2 or 3 donors were thawed and pooled in StemSpan ACF media (Stem Cell Technologies) containing 100 ng/mL SCF, 100 ng/mL FLT3LG and 20 ng/mL TPO (all from Miltenyi Biotec). After counting, cells were plated in 96 well round bottom suspension plates at 20,000 cells/well in the above media. Cells were allowed to recover overnight in these conditions in a 37° C. humidified incubator with 5% CO.sub.2.
(68) The next day cells were harvested from the plates counted, and seeded into 24 well Nanex plates (Compass Biomedical) or standard tissue culture treated plates at 2500 cells/well in 1 mL basal media (StemSpan ACF with 100 ng/mL SCF, 100 ng/mL FLT3LG and 20 ng/mL TPO) containing an HDAC inhibitor (for most experiments scriptaid was used at a concentration of 1 μM). An aliquot of cells was used for CFU analysis and another for flow cytometry analysis.
(69) After 3 days in culture the media was either replaced or supplemented with fresh media with the cytokines (SCF, TPO and FLT3LG) and the HDAC inhibitors (scriptaid 1 μM). Sixteen to twenty hours prior to harvest WR1065 was added at a concentration of 100 μM. After total of 5 days in expansion, cells are harvested, counted and analysed by flow cytometry.
(70) CFU Assays
(71) CFU assays were performed using the MethoCult Classic kit (Stem Cell Technologies) under manufacturer's instructions. Briefly, an aliquot of cells (either known number or volume) was mixed with 3 mL of MethoCult media and plated using a blunt needle and syringe into one well of a 6 well suspension plate. Plates were incubated for 2 weeks at 37° C. in a humidified incubator with 5% CO2 without media change. After 2 weeks, colonies were photographed under a dissecting microscope and counted. The proportion of colonies of different kinds was then scored and the proportion/sample was calculated.
(72) Flow Cytometry Assays
(73) Cells were harvested, washed and stained in 3% FBS in PBS using a panel of pre-conjugated antibodies. Cells were incubated with antibodies on ice for 30 min, then washed twice in 3% FBS in PBS and resuspended in 250 uL of above buffer before being analysed using a BD Canto II flow cytometer. [Antibody panel was as follows: 5 Lin custom cocktail (CD235a, CD4, CD10, CD11b and CD19) APC-Vio770, CD38-PE-Vio770, CD34-PerCP-Vio700, CD133-PE, CD45RA-VioBlue, CD49f-FITC, CD90-APC (all from Miltenyi Biotec).]
(74) Cell Counting
(75) Cell number was measured using flow cytometry with the aid of counting beads (CountBright beads Life Technologies).
(76) Results
(77)
(78) The fold expansion of the specific HSC-enriched population characterised by the phenotype Lin−, CD38-, CD34+, CD45RA−, CD133+, CD90+, CD49f+, is 20 fold higher in the combination treatment than in cytokines alone. Measurements of the number of cells with the phenotype (Lin−, CD38−, CD34+, CD45RA−, CD133+, CD90+, CD49f+) present at the end of the culture compared to those seeded at the beginning of the expansion can be compared and represented as a “fold expansion”. Comparison of the fold expansion shows cells treated with scriptaid have approximately 10 fold higher expansion than the cells grown in the basal media containing cytokines. The cells grown in the combination of scriptaid and WR1065 show a 20-fold higher expansion than the cells grown in the basal media containing cytokines (
Example 2 Expansion of Cells from Bone Marrow
(79) The expansion protocol, as defined above, was also performed on cells derived from bone marrow. Bone marrow cells show a 5 fold expansion of both total nucleated cells and CD34+ cells when cultured in the combination treatment. However, the HSC (characterised by the phenotype Lin−, CD38−, CD34+, CD45RA−, CD133+, CD90+, CD49f+) cell expansion is 17 times higher for the cells grown in scriptaid and WR1065 compared to that of the cells grown in basal media containing cytokines
Example 3 Expansion of Cells Derived from Peripheral Blood
(80) The expansion protocol, as defined above, was also performed on cells derived from peripheral blood. The overall fold expansion of cells derived from peripheral blood was lower than those of cells derived from umbilical cord blood. Peripheral blood cells show a 2-6 fold expansion of both total nucleated cells and CD34+ cells under all growth conditions. However the HSC (characterised by the phenotype Lin−, CD38−, CD34+, CD45RA−, CD133+, CD90+, CD49f+) cell expansion is 300 times higher in cells grown in scriptaid and WR1065 compared to cells grown in basal media with cytokines
Example 4 Expansion of Cells Using Class I HDAC Inhibitors RG2833 and RGFP966
(81) The expansion protocol as defined above was performed using the class I HDAC inhibitors RG2833 and RGFP966. RG2833 is selective for HDAC 1 and 3 and RGFP966 is selective for HDAC 3. Cells were expanded in media containing cytokines (basal), basal media containing either RG2833 or RGFP966, and basal media containing either RG2833 and WR1065 or RGFP966 and WR1065.
(82)
(83) These examples show that cells treated with the class I HDAC inhibitor and WR1065 show an increased fold expansion of the TNC compared to cells grown in basal media. The cells treated with the class I HDAC inhibitor and WR1065 also show an increased fold expansion of the HSC compartment above that seen in cells grown in basal media or treated with the class I HDAC inhibitor alone.
Example 5 Expansion of Cells Using Class IIa HDAC Inhibitor LMK235
(84) The expansion protocol as defined above was performed using a class IIa HDAC inhibitor LMK235. LMK235 is selective for HDAC 4 and 5.
Example 6 Expansion of Cells Using Class IIb HDAC Inhibitor Tubastatin A
(85) The expansion protocol as defined above was performed using a class IIb HDAC inhibitor Tubastatin A. Tubastatin A is selective for HDAC 6.
Example 7 Expansion of Cells Using a Broad Spectrum HDAC Inhibitors Sodium Phenylbutyrate and Quisinostat
(86) The expansion protocol as defined above was performed using the broad spectrum HDAC inhibitors sodium phenylbutyrate and quisinostat. Sodium phenylbutyrate is currently used to treat urea cycle disorders and quisinostat is currently in clinical trials for use in cancer. Cells were expanded in media containing cytokines (basal), basal media containing either sodium phenylbutyrate or quisinostat, and basal media containing either sodium phenylbutyrate and WR1065 or quisinostat and WR1065.
(87)
Example 8 Expansion of Cells from Individual Donors Using Scriptaid and WR1065
(88) The expansion protocol as defined above was performed of UCB cells from three separate donors, as well as a pooled sample of cells. The cells were expanded in media supplemented with cytokines (basal) media containing scriptaid and media containing scriptaid plus WR1065.
Example 9 Expansion of Cells Using Various Tissue Culture Plates
(89) The combination treatment of scriptaid and WR1065 enhances the expansion of HSCs cells regardless of the type of surface used to grow the cells. The use of scaffolds in cell culture is known to better mimic in vivo conditions and consequently provide a better niche for cells to grow. All previous examples were performed using Nanex plates which comprise a thin scaffold material made of PES electrospun fibres treated with a surface amination. Experiments were performed to show that treatment with scriptaid and WR1065 enhances the expansion of HSC when cells are cultured using standard tissue culture treated plates as well as Nanex plates. The cell expansion protocol was performed as described above and cells were seeded at the same densities onto 3 types of surfaces; Nanex scaffolds, TC treated Corning 24 well plates or suspension Greiner Bio 24 well plates. The fold expansion of HSCs for cells cultured in media supplemented with cytokines (basal) is significantly higher for cells grown in Nanex plates. However, fold expansion of HSC is significantly increased for cells treated with either scriptaid or the combination treatment when compared to cells cultured in basal media. This effect is similar across all surfaces used and the small differences are statistically non-significant.